Nitric oxide, produced in response to inflammatory cytokines or supplied exogenously, causes widespread cellular damage. Nonetheless, β cells can survive this onslaught and repair the damage if nitric oxide is removed from the cells before a critical threshold of damage is reached (8
). The mechanisms and participants responsible for this recovery process are only beginning to be elucidated. We showed previously that AMPK is transiently activated in a nitric oxide-dependent fashion in response to IL-1β or by the exogenous addition of nitric oxide using donors, and this activation is associated with improved metabolic function and an attenuation of β-cell death following nitric oxide treatment (34
). This is particularly important for β cells because proper metabolic function (oxidation of glucose and production of ATP) is essential for glucose-stimulated insulin secretion. While these previous studies outlined an important protective action of AMPK, the mechanisms responsible for nitric oxide-mediated AMPK activation in β cells were unknown.
To explore the mechanisms by which nitric oxide modifies AMPK signaling, we initially evaluated the effects of inhibitors and siRNA knockdown for each of the known AMPK kinases, LKB1, CaMKK, and TAK1. In all cases, inhibition or siRNA knockdown did not inhibit nitric oxide-stimulated AMPK activation, suggesting that each of these known AMPK kinases is dispensable () for the stimulatory actions of nitric oxide on AMPK. Nitric oxide has also been shown to activate ER stress responses in β cells, and the temporal nature of this activation correlates with the ability of nitric oxide to activate AMPK. Specifically, nitric oxide stimulation of AMPK phosphorylation correlates temporally with PERK and eIF2α phosphorylation, suggesting that ER stress may influence the activation of AMPK in response to nitric oxide. In examining this hypothesis, we made the unexpected observation that the IRE1 pathway regulates AMPK activation in response to nitric oxide. This signaling through AMPK leads to IRE1-dependent regulation of mTOR. Interestingly, this appears to be selective for nitric oxide, as cells lacking IRE1 respond normally to LKB1-mediated activation of AMPK (e.g., H2O2) (G). PERK, an eIF2α kinase, does not participate in the regulation of AMPK in response to nitric oxide, nor does the general induction of ER stress using classical activators tunicamycin, DTT, or thapsigargin (). These findings suggest a novel signaling role for IRE1 in the activation of AMPK in response to nitric oxide (). This signaling pathway is selective for nitric oxide, as classical activators of ER stress do not stimulate AMPK activation, and IRE1 deficiency does not lead to a general loss of AMPK activation ().
Fig. 7. Schematic diagram of nitric oxide-activated AMPK activation in β cells. Nitric oxide induces IRE1-dependent phosphorylation of AMPK and subsequent AMPK-dependent phosphorylation of Raptor and ACC. The AMPK signaling node connects IRE1 to mTORC1 (more ...)
Downstream of the IRE1-dependent activation of AMPK is the attenuation of mTOR signaling through the phosphorylation of Raptor. The ability of nitric oxide to modify the phosphorylation status of Raptor is mediated by AMPK, as adenovirus-mediated expression of a dominant negative mutant of AMPK attenuates nitric oxide-induced Raptor phosphorylation (D), consistent with previous findings (13
). In response to nitric oxide, AMPK, in an IRE1-dependent fashion, inhibits mTORC1 signaling, in part, through the phosphorylation of Raptor leading to the dephosphorylation and inactivation of S6K1. These results are consistent with the effects of AMPK activation and inhibition of mTORC1 signaling, in part through the phosphorylation of Raptor, leading to the dephosphorylation and inactivation of S6K1 in response to cell stress (13
). While we have focused on delineating the mechanism of AMPK activation in response to nitric oxide and the downstream influence on mTOR signaling in the context of Raptor, this is only one aspect of a complex signaling network altered by nitric oxide that influences mTOR signaling. In C, we show that nitric oxide reduces the activation-associated phosphorylation of Akt. When active, Akt inhibits the negative regulator tuberous sclerosis complex 2 (TSC2), and the inhibition of TSC2 promotes the activation of mTOR (26
). Thus, the ability of nitric oxide to remove the positive input from Akt would serve to further reduce mTOR activity. Additionally, Akt is a negative regulator, through inhibitory phosphorylation, of glycogen synthase kinase 3 (GSK3) (9
). GSK3 also phosphorylates TSC2, and this serves to inhibit mTOR; however, this requires a priming phosphorylation by AMPK on TSC2 (27
). Therefore, following exposure to nitric oxide, IRE1 may also signal to TSC2 through AMPK to reduce mTOR activity. One consequence of this signaling would be the inhibition of protein synthesis, and it is well known that nitric oxide is an effective inhibitor of protein synthesis in many cell types, including β cells (10
). Additional studies will clarify the role for each of these pathways in response to nitric oxide and the role of this regulation in cellular recovery and survival following nitric oxide-mediated damage.
The kinase(s) responsible for the direct phosphorylation of AMPK in response to nitric oxide has yet to be identified. The expression of a kinase-deficient IRE1α mutant in insulinoma cells did not prevent nitric oxide-induced AMPK activation, indicating that IRE1 does not directly phosphorylate AMPK. It is also unlikely that JNK phosphorylates AMPK under these conditions, as the knockout of IRE1α does not prevent nitric oxide-induced JNK phosphorylation (data not shown), consistent with nitric oxide-induced JNK activation's being dependent on sGC (46
). Our inhibitor and siRNA studies, summarized in , identified a number of broad signaling pathways that were not responsible for AMPK phosphorylation, including pathways dependent on calcium, mitochondrion permeability transition, ubiquitin, phosphatidylinositol-3-kinase (PI3K), HSP90, and others. Additionally, it is possible that nitric oxide-induced inhibition of metabolic enzymes such as cytochrome c
) is necessary to elevate AMP levels and promote the AMP-bound and phosphatase-resistant AMPK conformation (43
Cells may modify the mechanism of AMPK activation depending on the level of nitric oxide and/or cell type. At low concentrations of nitric oxide, replicating those produced by endothelial NOS (eNOS), AMPK is activated by CaMKK in a guanylate cyclase-dependent fashion in endothelial cells (61
). However, in response to high levels of nitric oxide, replicating those produced by iNOS, AMPK activation is unaffected by CaMKK inhibition with STO-609, guanylate cyclase inhibition with ODQ, and calcium chelation with BAPTA-AM (). While these findings suggest concentration-dependent mechanisms of action of nitric oxide, lower concentrations of nitric oxide (DEANO, 100 μM) also stimulate AMPK phosphorylation in β cells in an IRE1-dependent fashion (B). These findings suggest that the role of IRE1α in the regulation of AMPK by nitric oxide may be cell type dependent. Somewhat unique to pancreatic β cells is the efficient folding, trafficking, and secretory capacity that is needed for the synthesis and secretion of insulin. In addition, there is a strict dependence of β cells on oxidative metabolism (glucose to CO2
) for function (insulin secretion) and, because of this secretory demand, β cells have an adaptive response to oxidative stress that is associated with UPR induction (48
). Thus, it seems physiologically plausible to couple the induction of protective responses to cellular stress (IRE1α and the UPR) with regulators of oxidative capacity (AMPK) to provide a mechanism by which β cells coordinate the regulation of oxidative metabolism with the response to cellular stress as mechanisms to afford protection from nitric oxide. In addition, the concentration- and cell type-selective nature of these actions could also suggest that the mechanism of nitric oxide-induced AMPK activation is dependent on the physiological context, e.g., regulation of vascular relaxation (eNOS) versus cell defense in response to inflammation (iNOS) and injury.
The finding that ER stress alone is not sufficient to stimulate AMPK activation () suggests that IRE1 may be differentially activated in response to nitric oxide compared to ER stress-inducing agents such as tunicamycin and thapsigargin. Whereas tunicamycin stimulates both kinase and RNase activation of IRE1 (60
), stimuli such as nitric oxide and quercetin may preferentially activate IRE1 RNase activity. Quercetin has been shown to activate IRE1 RNase activity independent of kinase activation (55
), while nitric oxide induces RNase activation, but the impact on kinase activation is unknown and requires further investigation. Our findings that quercetin induces IRE1-dependent activation of AMPK and that cells expressing kinase-deficient IRE1 have enhanced nitric oxide-induced AMPK activation suggest that IRE1 RNase activation in response to these agents participates in a pathway leading to AMPK phosphorylation and that this response may be attenuated by IRE1 kinase activation. This is in line with the recent finding that the kinase and RNase activities of IRE1 can differentially impact cell fate (14
). Thus, the RNase-dependent activation of AMPK may promote adaptive responses through upregulation of genes such as PGC-1α, while IRE1 kinase activation may suppress this response to promote elimination of irreparably damaged cells.
From an evolutionary standpoint, it is logical for IRE1 to play a regulatory role in the activation of AMPK signaling pathways. Both of these highly conserved proteins are responsive to cellular stress, and once active, they promote the restoration of cellular homeostasis and adaptation to environmental changes (15
). Additionally, ER stress has newly found roles in the regulation of lipid metabolism and gluconeogenesis (41
) pathways that have long been known to be regulated by AMPK (16
). The IRE1-AMPK pathway could also provide a mechanism to couple the ER stress pathway with the regulation of metabolism. This form of regulation may be critical for cell survival or the loss of cell viability in response to stress. By coupling these pathways, β cells would have an exquisite level of control over rapid posttranslational modifications and long-term adaptive responses through regulation of gene expression.